Due to the limited availability of suitable livers for transplantation and the increasing use of suboptimal donor livers, which are more prone to ischemia-reperfusion injury (IRI), there is an urgent need for novel graft-ameliorating therapies. The promising capacities of (liver-derived) mesenchymal stromal cells ((L-)MSC) in mitigating IRI and their integration with hypothermic oxygenated machine perfusion (HOPE) present a novel paradigm for augmenting graft quality. However, still little research exists into the effects on the liver of (L-)MSC administered during HOPE. Moreover, human livers for research are scarce and, therefore, studies to show safety and efficacy are challenging. To address these challenges, this study investigated the use of porcine precision-cut liver slices (PCLS) as a tool to model the administration of MSC during HOPE. First, optimisation of PCLS procurement and culture was undertaken, exploring methods such as the use of well inserts and different culture media to enhance the retained structural integrity of the slices. Subsequently, a 24-hour co-culture experiment of porcine PCLS (n=3 livers, n=135 PCLS) and L-MSC was conducted, where L-MSC were added at the beginning of a two-hour hypothermic oxygenated culture. The findings of this experiment demonstrated the ability of MSC to adhere to the liver tissue during the two hours in a hypothermic oxygenated environment. Additionally, the effect of MSC on PCLS viability, morphology, cell death, regeneration, liver injury markers, functionality, and gene expression profiles was investigated. While no significant differences between co-cultured and control PCLS were observed, there was a clear trend towards increased inflammation, cell damage, and cell death in the co-cultured slices. Overall, this research contributes to the understanding of the potential implications of (L-)MSC administration during hypothermic oxygenated perfusion and highlights the utility of PCLS as an intermediate experimental model for studying transplant-related IRI.

Cholangiocarcinoma (CCA) is a rare but aggressive type of primary liver cancer with a dismal prognosis. CCA commonly metastasizes to the lungs and lymph nodes, significantly reducing overall survival. However, a mechanistic understanding of how CCA invades these metastatic sites remains lacking. This is partly due to the failure of current models to recapitulate the complexity of tissue-specific environments for metastatic CCA. Patient-derived tumor organoids are promising models for studying cancer in vitro, including CCA. However, organoids are frequently cultured in basement membrane extract (BME), which does not represent the native microenvironment. Decellularized scaffolds can serve as a culture environment for investigating tumor-extracellular matrix (ECM) interactions. Nonetheless, decellularized scaffolds are often utilized in conjunction with cancer cell lines, lacking critical characteristics of a growing tumor in vivo. Therefore, this study aims to decellularize and characterize human-derived lung (n=3) and lymph nodes (n=17), and recellularize these with patient-derived CCA organoids (CCAOs) (n=3) to establish an in vitro model for studying the interaction between epithelial tumor cells and the microenvironment of the metastatic site. Decellularization resulted in acellular scaffolds with preserved ECM components. Local ECM heterogeneity was shown by the macro- and micro-scale mechanical properties as determined by rheology and micro-indentation. The CCAOs showed adherence and growth when combining them with the decellularized lung ECM (dLECM). The metabolic activity of CCAOs was diminished when cultured in dLECM compared to those cultured in BME, and was dictated both by the organoid line and the dLECM donor. Distinct CCAO gene expression profiles were observed between the two culture environments (BME and dLECM). This elucidates the effect of the culture environment on the behavior of CCAOs in an in vitro model. In conclusion, the convergence of CCAOs with their organ-specific location of metastasis obtained by decellularization, provides a valuable tool for integrating the ECM of the metastatic location in an in vitro model for the mechanistic study of cancer metastasis.

To this day, liver transplantation is the only effective treatment for end-stage liver failure. Unfortunately, the scarcity of liver donors leads to waitlist mortality, pushing the need for alternative treatment options. In vitro models are essential tools in research on alternatives for transplantation. One of the promising models is the hepatobiliary organoid model because these three-dimensional cultures model (elements of) the native tissue structure and function. However, a limitation of these models is that they currently resemble only one liver cell type. The aim of this research was therefore to combine different cell types, i.e., biliary organoids and mesenchymal stromal cells (MSCs), in one culture model.

The organoids were obtained from liver-biopsy-derived intrahepatic biliary epithelium and are therefore named intrahepatic cholangiocyte organoids (ICOs). The liver-specific MSCs (L-MSCs) were isolated from perfusion fluid of donor livers collected at liver transplantation procedures. Decellularized liver tissue was used to include extracellular matrix (ECM) in the co-culture models. To assess the most optimal culture conditions, three experimental setups were tested. In the first setup, ICOs were cultured in different concentrations of L-MSC derived conditioned medium (CM) in commercially available basement membrane extract (BME) (1A). This setup was also used to differentiated the ICOs towards hepatocytes (1B). In the second setup, various concentrations of ICOs and L-MSCs were cultured in an indirect (2A) and direct (2B) BME model. The third setup included liver-derived ECM to replace the BME. Cells were cultured similar to model 2B (3A) and ICOs were added after a pre-culture of L-MSCs (3B). The morphology of both cell types, the visible interaction between cell types, and the expression of hepatocyte, cholangiocyte, MSC and proliferation markers were analyzed.

The effect of L-MSCs and L-MSC-derived CM on ICO formation, morphology, and growth patterns, was small. Direct cell-cell contact was rare in the BME co-cultures, but instances of close contact between two cell types were observed in both the indirect (2A) and direct (2B) setups. In the ECM models (3A-B), ICO cells formed polarized monolayers that pushed the L-MSCs aside, indicating that the cell types did not mix well. ICOs expressed the cholangiocyte markers cytokeratin (CK) 7 and 19 in both mono- and co-cultures, whereas L-MSCs were most likely CK7 and CK19 negative. Variation in gene expression levels of hepatocyte, cholangiocyte, MSC, and proliferation markers was observed, but no statistically significant differences were found between conditions. Nevertheless, the down-regulatory effect of CM on the expression of hepatocyte markers in differentiated ICOs showed a consistent trend. Additionally, the presence of L-MSCs resulted in a higher mean expression of MSC markers CD90, CD105 and Vimentin, and proliferation marker KI-67.

Although no clear effects of L-MSCs on ICO growth were observed in this study, the created co-culture models form a promising base for future research. The models include adult human liver-derived components and are therefore a step towards the reconstruction of the hepatic microenvironment. In addition, if other (liver-derived) cell types were to be introduced to the co-culture models, L-MSCs have the potential to enhance the cell function of these new cells. Last, the models can be used as a base for future ICO co-culture studies, since the L-MSCs can be replaced by other cell types.

Cholangiocarcinoma (CCA) is a highly aggressive biliary tumor with a poor prognosis and limited treatment options. Various critical aspects of CCA development remain unclear. Lately, the dynamic process of epithelial-mesenchymal transition (EMT) was highlighted to play a crucial role in the fundamental mechanism of metastatic dissemination. However, in vitro EMT studies are mainly limited to conventional 2D models which lack in vivo tumor physiology. Recently, 3D organoid culture methods provide us new model possibilities. Therefore, our research aim was to establish a representative in vitro organoid-based epithelial-mesenchymal transition model in cholangiocarcinoma including its in vivo pathophysiology and tumor microenvironment. This study focusses on three different aspects of the tumor microenvironment regarding EMT activation in CCA organoids: 1.The effects of EMT promoting growth factors and culture media on EMT activation. Various growth factors are highlighted to play a crucial role in EMT activation in different cancer types. To study EMT activation in CCA, various growth factors were added to CCA organoids. We observed round shaped organoid structures consisting of single layered epithelium for all conditions. Additionally, gene expression levels of EMT related markers were increased for specific growth factor conditions. The use of various culture media combined with transforming growth factor β1 (TGF-β1) showed the most substantial differences in mRNA expression of EMT markers in branching medium (BM). Based on these results, specific growth factors and BM were used to find the optimal culture conditions to induce EMT. Although we did not find significant morphological differences between the various conditions, altered gene expression levels of EMT markers were observed after TGF-β1 and tumor necrosis factor alpha (TNF-α) treatment in BM. 2.The effect of the tumor extracellular matrix on EMT activation. The tumor extracellular matrix (ECM) is an important component of the tumor microenvironment that stimulates the malignant behavior of surrounding cells. The combination of CCA organoids and patient-derived ECM resulted in morphological changes. Cells with a polygonal shape adopted an irregular shape after TGF-β1 treatment. On top of that, our data revealed that patient-specific ECM and CCA organoids altered the gene expression of EMT related markers. 3.The effect of the interstitial fluid flow on EMT activation. The use of a microfluidic platform enabled us to study the effect of fluid flow on EMT in vitro. Cells formed organoid structures in the absence and the presence of a fluid flow. However, cells only migrated to the surrounding culture medium due to the presence of a fluid flow. When cells were seeded directly into the chip, cells formed a tube structure with protrusions growing outwards. The protrusions growth was accelerated by the addition of TGF-β1 and led to increased cell migration. Additionally, fluid flow conditions resulted in altered gene expression levels of EMT related markers. Our data suggest that fluid flow could potentially stimulate EMT in CCA organoids. Overall, our findings have demonstrated that growth factors, native ECM and interstitial fluid flow could be used to induce EMT in vitro. Indicating that the tumor microenvironment plays a crucial role in EMT and tumor progression. Our novel in vitro CCA organoid-based EMT model provides a foundation towards a more representative EMT in vitro model to unravel the underlying mechanism of CCA and its progression.